1. Field of the Invention
This invention pertains to the administration of lysostaphin for the purpose of treatment of staphylococcus infection in mammals, including humans, as well as pharmaceutical preparations used in said treatment. This invention also pertains to methods of addressing particular disease conditions, including staphylococcal endocarditis; staphylococcal bacteremia; and staphylococcal infection of kidneys, lungs, skin, bone, burns, wounds and prosthetic devices. The invention embraces the use of lysostaphin broadly, including not only wild type lysostaphin but recombinant lysostaphin; lysostaphin variants with amino acid sequences varying from the published `natural sequence` of the mature peptide (U.S. Pat. No. 4,931,390) due to genetic mutations (such as substitutions, additions, deletions), post-translational processing, or genetic engineering, chimeric fusion proteins and the like; or a combination of these kinds of variations.
2. Background of the Prior Art
Lysostaphin is an enzyme, first identified in Staphylococcus simulans (formerly known as S. staphylolyticus), which has antimicrobial activity by virtue of its proteolytic activity on glycine-containing bridges in the cell wall peptidoglycan of bacteria (Zygmunt, et al., Progr. Drug Res. 16:309-333 (1972)). In vitro, lysostaphin is particularly active against Staphylococcus aureus, because the cell wall bridges of this species contain a high proportion of glycine, although activity against other species of staphylococci has been demonstrated (Ibid.).
The activity of lysostaphin has also been explored in animal infection models. For the purposes of this discussion, the results of intraperitoneal treatment after intraperitoneal infection will not be considered; this experimental design is similar to an in vitro experiment. In intraperitoneal infection models there have been two reports of survival of 50% of treated mice after single or multiple subcutaneous administrations of a total of approximately 1 mg/kg of a lysostaphin preparation (Schuhardt, et al., J. Bacteriol. 88:815-816 (1964); Harrison, et al., Can. J. Microbiol. 13:93-97 (1967)). A total dosage of 6 mg/kg was reported to protect 100% of the mice in one of these studies (Harrison, et al., Ibid.). The virulence of the bacterial challenge used in both studies appears to be quite low, as the untreated infected mice did not all die within a short period of time.
Several experiments used a mouse subacute model measuring the bacterial load in the kidneys after infection with the Giorgio strain of S. aureus (Dixon, et al., Yale J. Biol. Med. 41:62-68 (1968); Schaffner, et al., Yale J. Biol. Med. 39:230-244 (1967); Harrison, et al., J. Bacteriol. 93:520-524 (1967)). When a lysostaphin preparation was administered intravenously within 6 hours after infection, significant reductions in the numbers of bacteria in the kidneys were observed with dosages of 1.5 mg/kg or higher. However, established infections were more refractory; only modest reductions in the numbers of bacteria were seen when treatment was withheld for 24 hours or longer, even with dosages of 125 or 250 mg/kg of a lysostaphin preparation. The effect of multiple treatments was not studied.
A single study, (Goldberg, et al., Antimicrob. Ag. Chemother. 1967:45-53 (1967)), employed a limited number of dogs in an unusual endocarditis model. The dog model has not been further developed. The Goldberg, et al., experiment was not comparative, and is therefore of limited utility in assessment of the administration of lysostaphin. However, high dosages of lysostaphin (at least 50 mg/kg/treatment) were only moderately effective, as judged by the health of the dogs and by the extent of reduction in the number of bacteria in the heart valves and kidneys.
Limited human trials were conducted aimed at eradication of nasal carriage of S. aureus by topical application of lysostaphin to the nares (Martin, et al., J. Lab. Clin. Med. 70:1-8 (1967); Martin, et al., J. Lab. Clin. Med. 71:791-797 (1968); Quickel, et al., Appl. Microbiol. 22:446-450 (1971)). Nasal carriage is not in itself a disease state. It does constitute a risk factor for infection of patients treated by colonized health care professionals or for self-infection in the case of a colonized patient.
The art reports treatment of one very ill human patient with a single dose of parenterally administered lysostaphin, followed by an antibiotic, gentamicin, three days later. The patient died, but did exhibit a reduction in bacteremia (Stark, et al., N. Engl. J. Med. 291:239-240 (1977)).
Immunogenic phenomena observed during the course of the animal and human studies, were noted as a great concern. Contamination of the lysostaphin preparations with extraneous substances may have been responsible for at least some of these phenomena.
No further development of the enzyme as a therapeutic agent occurred, given the lack of desired effectiveness in the studies discussed. This may have been further due to the difficulty in producing and purifying lysostaphin.
The staphylococcal gene for lysostaphin has been sequenced and cloned (U.S. Pat. No. 4,931,390). Lysostaphin for use as a laboratory reagent has been produced by fermentation of a non-pathogenic recombinant strain of Bacillus sphaericus, from which it is readily purified.
Although this previous art did not teach that lysostaphin is highly effective in clearing established infections from various organs in animal models, more recently it has been demonstrated that a regimen of multiple, relatively low, doses of lysostaphin was surprisingly effective in curing experimental endocarditis in rabbits caused by methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin intermediate susceptible S. aureus (VISA)(U.S. patent application Ser. No. 09/120,030, filed Jul. 21, 1998; Climo, et al., Antimicrob. Agents Chemother. 42:1355-1360 (1998).) The good tolerability of lysostaphin in the rabbit model suggests that a multiple dose regimen of lysostaphin, alone or in combination with other antibiotics, may be practicable in treating human disease. However, it remains an object of those of skill in the art to develop the most tolerable and most effective means of using lysostaphin to treat human staphylococcal disease.
Conventional administration of antibiotics typically involves an extended course of low to medium level dosage. Particularly resistant infections, such as osteomyelitis may require treatment over many weeks, or even months. Other staphylococcal infections may be treated in a shorter time period, but nonetheless, typically extend over 10-14 days. In severe infections, that present extreme life-threatening conditions, such as endocarditis, even the most aggressive antibiotic therapy typically contemplates a period of administration of at least several weeks.
Before the infection is completely cleared, organ damage often results from infections with highly virulent bacteria, such as S. aureus. Additionally, staphylococcal infections, particularly with S. aureus, can evolve from an initial, relatively benign infection (such as low-grade bacteremia or skin infection) to a deep-seated single or multiple organ infection, including endocarditis. These events may occur despite using the best available antibiotics, because antibiotic action is not rapid enough.
Furthermore, it is known that even the safest drugs can have undesired side effects. Although lysostaphin has thus far not been shown to have adverse effects in animal models, other protein drugs are known to cause immunogenic reactions in humans after prolonged treatment. It is also possible that prolonged treatment will induce the production of neutralizing antibodies, which would progressively reduce the effectiveness of the protein therapeutic.
Accordingly, it remains an object of those of ordinary skill in the art to provide an effective treatment regimen that effectively eradicates even significant, established staphylococcal infections and prevents the development of serious infections, that contemplates administration over a very short period.